| Objective:To study the protection induced by transient limb ischemic conditioning during myocardial ischemic period against myocardial ischemia reperfusion (I/R) injury of rats.Method:Healthy male Wistar rats were administered. The heart was exposed through lower sternal incision and the left anterior descending coronary artery was ligated by snipknot to establish the ischemia/reperfusion model. A catheter was put into the left ventricle from the apex of heart to trace the pressure of left ventricle and the pressure change index while electrocardiogram was monitored. Rats were assigned randomly into seven groups. (1)Sham group (n=8):rats were threaded a silk suture under the left coronary artery anterior descending (LAD) and laid up through out the experiment. (2)IR group (n=12):rats were subjected to 30 min LAD occlusion followed by 180 min reperfusion. (3) RP group (transient limb ischemic conditioning during myocardial ischemia period, remote preconditioning group) (n=10):rats were subjected to three episodes of 5 min double hind limbs occlusion followed by 5 min reperfusion during 30 min myocardial ischemia period which follower by 180 min reperfusion. (4) RIPC group (remote ischemia preconditioning group) (n=11):rats were subjected to three episodes of 5 min double hind limbs occlusion followed by 5 min reperfusion before 30 min myocardial ischemia period which follower by 180 min reperfusion. (5) RI-PostC group (remote ischemia post-conditioning group) (n=11):rats were subjected to three episodes of 5 min double hind limbs occlusion followed by 5 min reperfusion at the very beginning of reperfusion during 30 min myocardial ischemia and 180 min reperfusion period. (6)GL group (Glibenclamide group) (n=11):rats were subjected to three episodes of 5 min double hind limbs occlusion followed by 5 min reperfusion during 30 min myocardial ischemia period while glibenclamide was injected intraperitoneally dosed with 5 mg/kg which also follower by 180 min reperfusion. (7)CS group (Cyclosporin A group) (n=10):rats were subjected to 30 min LAD occlusion followed by 180 min reperfusion while CsA was injected into the left ventricle from the catheter dosed with 10 mg/kg.The myocardial protection induced by RP against IRI was evaluated by reperfusion arrhythmia (RA), cardiac contractility, marker of myocardial injury, infarct size, cardiocyte death, expression of apopto sis-relevant proteins. The scores of ischemia arrhythmia and reperfusion arrhythmia, the left ventricular developing pressure, the maximal change index of LV (±dp/dtmax) and recovery percentage of maximal change index were calculated. Serum CK (creatine kinase) and c-TNT (cardiac troponin T) were tested by ABC ELISA. Myocardial definite change of tissue morphology was inspected by HE staining. Myocardial infarct size was measured by TTC staining technique. The expression of apoptosis-relevant proteins, Bcl-2 and Bax, were tested by histochemistry immunity method, and caspase-3 was tested by western blot.Results:1. Evaluation of animal model.Compare IR group to Sham group, the ST segment of ischemia period was significant elevated(P<0.01), the scores of ischemia and reperfusion arrhythmia were significant increased(P<0.05), the maximum systolic pressure of left ventricular and the idp/dtmax at the reperfusion starting point were significantly decreased, and the serum c-TNT was significantly higher (P<0.05).2. Influence on RA, cardiac contractility and infarct size of remote perconditioning against rats'ischemia reperfusion injury.The RA score of early reperfusion phase, change index and maximal pressure of left ventricle at end point of RP group all fell in between IR group and sham group. The arrhythmic score of RP group was (1.50±0.97) and the score of IR group was (2.33±0.71). The difference between them was statistically significant (P=0.049). There was no statistically significant difference on change index and maximal pressure of left ventricle between IR group and RP group. Difference of CK among three groups was not statistically significant. While the c-TNT of IR group was significant higher than that of sham group (P=0.021), there was no statistically significant difference between RP and IR group or between RP and sham group. There was no difference of ischemia size among three groups, while infarct sizeof RP group was significant smaller than that of IR group (P<0.01).3. Influence on cardiocyte apoptosis against rats'myocardial IRI.Compared to IR group, the percentage of cardiocyte of RP group which expressed Bax was statistically significantly lower (P<0.05), while the percentage of Bcl-2 was not (P>0.05). The rate of Bcl-2/Bax was statistically significantly higher (P<0.05). There was no significant difference on expression of caspase-3 between RP and IR group (P>0.05).4. Comparison among RP, RIPC and R1-PostC induced protection against rats' myocardial IRI.RA score of RP group was smaller than IR group (P<0.05) but it of RIPC and Rl-PostC group had no statistically significant difference compared to both RP and IR groups. There were no statistically significant difference of hemodynamics data between RP group and IR group. Compared to IR group, LVDPmin of RIPC group were higher at reperfusion 30 min (P<0.05) and-dp/dtmax of RI-PostC group was lower at reperfusion 0 min (P<0.05). Other data difference was not statistically significant. Difference of serum CK and c-TNT among four groups was not statistically significant. There was no difference of ischemia size among four groups, while the infarct size of RP, RIPC and RI-PostC group was statistically significantly smaller than IR group (P<0.01). The infarct size percentage of RP group was (15.27±5.19), it of RIPC group was (14.53±3.45), and it of RI-PostC group was (19.84±5.85), while the size percentage of IR group was (34.47±7.13). There was no difference of infarct size among three conditioned groups.Expression of Bcl-2 in three groups was higher than it of IR group. Bcl-2 of RI-PostC group was statistically significant higher than it of RP and IR groups while that of RIPC group was higher than that of IR group (P<0.05). Expression of Bax in three groups was similar and those of RP and RIPC group were statistically significant lower than it of IR group (P<0.05). The Bcl-2/Bax positive unit rate of IR group was lower than RP, RIPC and R1-PostC group. The difference between IR and RP group was statistically significant (P<0.05). There was no significant difference on Bcl-2, Bax and Bcl-2/Bax among three conditioned groups. There was also no significant difference on caspase-3 among these four groups.5. Impact of glibenclamide on remote perconditioning induced myocardial protection.RA scores (P<0.05) of GL group was lower than IR group. The LVSPmax and±dp/dtmax were higher than IR group (P<0.05) at reperfusion 0 min. But there were no significant difference of arrhythmia scores and cardiac contractility compared to RP group. Serum c-TNT of GL group was lower than RP and IR group (P<0.05). The infarct size of GL group fell in between RP group and IR group, the difference compared to RP group was statistically significant (P<0.05) while it compared to IR group was not.Expression of Bcl-2 in RP group and GL group were higher than IR group and there was statistically significant difference between GL and IR group. Expression of Bax in GL group fell in between RP group and IR group, while no statistically significant difference was found compared to RP and IR group. The difference of Bcl-2/Bax positive unit rate wasn't significant. Expression of caspase-3 in GL group was higher than both IR and RP group (P<0.05).6. Influence of CsA applied during myocardial ischemia period against rats' myocardial IRI.RA scores (P<0.05) of CS group was lower than IR group, while no significant difference was found compared to RP group. Compared to IR group, LVDPmin was higher at reperfusion 0 and 60 min; LVSPmax was lower at reperfusion 120 min; recovery percentage of±dp/dtmax was lower from reperfusion 30 min to 180 min(P<0.05).Compared to RP group, LVSPmax,-dp/dtmax at reperfusion 60 min, LVSPmax at 120 min and±dp/dtmax at 180 min of CS group were lower; recovery percentage of-dp/dtmax from 30 to 180 min and dp/dtmax from 30 to 60 min were lower; LVDP at 60 min was also lower than RP group (P<0.05).Serum c-TNT of CS group was lower than RP and IR group but not statistically significant. The infarct size of CS group fell in between RP group and IR group, the difference between CS and IR group was statistically significant (P<0.05). Expression of Bcl-2 in RP group and CS group were higher than IR group but there was no statistically significant difference. Expression of Bax in CS group fell in between RP group and IR group, while no statistically significant difference was found. The sequence of Bcl-2/Bax positive unit was IR group, CS group and RP group. The difference compared to RP group was significant (P<0.05). Expression of caspase-3 in CS group was higher than IR group (P<0.05), but had no significant difference with RP group.Conclusions:1.The method of establish rat myocardial ischemia/reperfusion model througu lower sternal incision is simple and convenient, and the experiment results are reliable.2.RP method can reduce rats'myocardial IRI. It was found to alleviate RA, decrease infarct size, improve myocardial morphological lesion, and protect left ventricle contractility. It maybe the mechanism that RP inhibits IRI induced cardiocyte apoptosis by decreasi ng expression of Bax, the pro-apoptotc protein.3.The three methods of RP, RIPC and RI-PostC executed by transient limb ischemia conditioning all have equal myocardial protection ability against IRI, displayed as decreased infarct size, inhibited cardiocyte apoptosis. RP method is even better on alleviating RA.4.Glibenclamide injected intraperitoneally can alleviate ischemia arrhythmia score and partly counteract myocardial protection induced by RP. CsA injected into heart chamber can partly simulate RP method and induce some protection. But its hiding injury can abolish its benign effect. The most important effecters of RP induced endogenous myocardial protection is mKATP and mPTP.
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